Project 1: Vascularization and Growth of Human Myocardial Grafts The goal of this project is to use lessons from cardiovascular development and stem ceU biology to create functional myocardium. Pluripotent human stem cells can now be directed to myocardial fates, yielding human myocardial cells that survive in the infarct, improve cardiac function and should be immunologically compatible with the host. Our central hypothesis is that significant growth and function of these grafts requires co-development of a coronary vascular supply, and that augmented vascularization will promote remuscularization. Three specific aims are proposed. Aim 1 builds on observations from tissue engineering, demonstrating that mouse fibroblasts and HUVECs cooperate with human cardiomyocytes in the formation of vascularized myocardial tissue. We will identify clinically relevant sources of mesenchymal cells and endothelium by screening MSC clones and endothelium derived from hESCs and EPCs, first in a cardiac tissue engineering and then in infarcted hearts. Aim 2 explores the suitability of a novel cardiovascular progenitor cell derived from hESCs for cardiac repair. This multipotent mesodermal progenitor is identified by expression of KDR (VEGFR2) and is capable of generating human cardiomyocytes, endothelium and smooth muscle cells. We will test the ability of KDR-i- cells to form human myocardium and a human coronary circulation, which we predict will enhance myocardial graft size, myocardial blood flow and contractile function in the infarcted heart when compared to cardiomyocytes-only. Aim 3 will investigate the mechanism through which cardiac grafts induce formation of collateral arteries from host coronaries, exploring the role of hedgehog signaling and the monocyte in promoting arterialization. These experiments address key questions regarding mechanisms of cardiac repair and are the first to systematically address formation of a human coronary circulation in combination with new myocardium.